Modeling gap junction beta 2 gene-related deafness with human iPSC

Abstract

There are >120 forms of non-syndromic deafness associated with identified genetic loci. In particular, mutation of the gap junction beta 2 gene (GJB2), which encodes connexin (CX)26 protein, is the most frequent cause of hereditary deafness worldwide. We previously described an induction method to develop functional CX26 gap junction-forming cells from mouse-induced pluripotent stem cells (iPSCs) and generated in vitro models for GJB2-related deafness. However, functional CX26 gap junction-forming cells derived from human iPSCs or embryonic stem cells (ESCs) have not yet been reported. In this study, we generated human iPSC-derived functional CX26 gap junction-forming cells (iCX26GJCs), which have the characteristics of cochlear supporting cells. These iCX26GJCs had gap junction plaque-like formations at cell–cell borders and co-expressed several markers that are expressed in cochlear supporting cells. Furthermore, we generated iCX26GJCs derived from iPSCs from two patients with the most common GJB2 mutation in Asia, and these cells reproduced the pathology of GJB2-related deafness. These in vitro models may be useful for establishing optimal therapies and drug screening for various mutations in GJB2-related deafness.

Induced Pluripotent Stem Cells, a Stepping Stone to In Vitro Human Models of Hearing Loss

“…A number of these carry mutations in the Gap Junction Beta 2 (GJB 2) gene that codes for the CX26 protein and is associated with over half of the cases of hereditary HL in the world with more than 340 pathogenic variants [ 68 , 89 , 91 , 108 , 109 ]. Fukunaga and colleagues (2021) have recently reported that absence of the CX26 protein in gap junction-forming inner ear SC-like cells generated from patients’ hiPSCs results in deficient gap junction-mediated intercellular communication, associated with shortened gap junction plaques in those cells [ 37 ]; in this case, the authors have reported similar findings to those obtained in GJB 2 mutant mice [ 110 ]. hiPSC lines have been created from patients who carry mutations in another gene that is expressed in cochlear SCs, the Transmembrane Protein 43 (TMEM 43) gene [ 111 ].…”

“…Most of the work that has been carried out to establish in vitro models of inner ear development has focused on the two main sensory cells in the cochlea, the hair cells (HCs) and the spiral ganglion neurons (SGNs). However, protocols are now being developed to generate other otic cell types [ 36 , 37 ]. Additionally, the last years have seen a surge in the number of studies that are conducted on recently developed organoid models, 3D cultures that harbour different types of otic cells and that more closely mimic the in vivo situation.…”

“…The common marmoset, also amenable to genetic modification, has thus been proposed as a more predictive model of human cochlear development and pathology [ 21 , 22 ]. In addition, hiPSC lines are being generated from somatic cells obtained from patients with HL [ 89 , 90 , 91 , 92 , 93 ] and subsequently differentiated towards a range of different otic cell types [ 37 , 59 ], thus providing human cell-based models that overcome the limitations imposed by the complex anatomy of the inner ear and the lack of human tissue; very importantly, these cultures retain the genetic backgrounds and the mutations present in patients with hearing deficits, constituting an ideal substrate for studies on the molecular mechanisms that lead to syndromic or non-syndromic HL and the validation of therapeutic targets through the creation of isogenic control cell lines [ 59 , 88 , 94 ]. Additionally, they provide the means to model mutations within large non-coding chromosomic regions that would otherwise be very difficult to replicate.…”

Application of Human Stem Cells to Model Genetic Sensorineural Hearing Loss and Meniere Disease

“…The autosomal recessive non-syndromic SNHL is commonly caused by mutations in GJB2 gene which encodes for connexin 26 (CX26) [105]. An in vitro model for the homozygous 235delC mutation in GJB2 has been developed from hPSC to develop a therapy for deafness [106] (Figure 2). Moreover, we have started to generate inner ear organoids from the hiPSC-MD model derived from a patient with mutations in DTNA and FAM136A genes for studying the development of the inner ear in this patient as well as how mutations found in DTNA and FAM136A affect the development and functionality of the system itself when sensory organs mature [75,76].…”

“…The major achievements in disease modeling using a hiPSC-derived inner ear for genetic SNHL include the genes MYO7A, MYO15, and MERRF in HCs [70,89,90], and GJB2 and SLC26A4 (Pendred syndrome) in SCs [106,[123][124][125] (Table 2). These studies have defined the molecular mechanisms involving each gene and showed the cellular effects of each mutation.…”